Methods and reagents for protease inhibition

ABSTRACT

There is provided a protease inhibitor and a method of inhibiting a protease selected from the group consisting of thrombin, chymotrypsin and neuropsin, by contacting the protease with an effective amount of a member of the phosphoethanolamine binding protein (PEBP) family.

TECHNICAL FIELD

The present invention relates to proteases inhibitors, useful in thefields of protein chemistry, thrombogenesis, cancer and neurobiology.

INTRODUCTION

Serine proteases are involved in many processes during tissuedevelopment and homeostasis. Among other functions they degradecomponents of the extracellular matrix to allow outgrowth of neuronalprocesses (Monard, D. (1988) Trends Neurosci. 11, 541–544) or cellmigration (Seeds, N. W., Basham, M. E., and Haffke, S. P. (1999) Proc.Natl. Acad. Sci. U. S. A. 96, 4118–14123), they promote cell death(Tsirka, S. E., Rogove, A. D., Bugge, T. H., Degen, J. L., andStrickland, S. (1997) J. Neurosci. 17, 543–552) and act as mitogenic orsurvival factors (Vaughan, P. J., Pike, C. J., Cotman, C. W., andCunningham, D. D. (1995) J. Neurosci. 15, 5389–5401). The activity ofthe proteases is regulated by their cognate inhibitors, which have toact in an accurately balanced fashion to ensure normal development andhomeostasis. Disturbances of this balance in the nervous system havebeen proposed to be involved in pathological disorders such asAlzheimer's disease (Turgeon, V. L. and Houenou, L. J. (1997) Brain Res.Brain Res. Rev. 25, 85–95; Wagner S L, Geddes J W, Cotman C W, Lau A L,Gurwitz D, Isackson P J, and Cunningham D D (1989) Proc Natl Acad Sci US A 86 (21):8284-8).

To date, several serine proteases have been detected in the centralnervous system, including tissue type plasminogen activator (t-PA),chymotrypsin, neuropsin, elastase, and thrombin (Davies, B. J., Pickard,B. S., Steel, M., Morris, R. G., and Lathe, R. (1998) J. Biol. Chem.273, 23004–23011). Many serine protease inhibitors regulating theactivity of these proteases are found in the brain. However, theprotease nexin-1 (PN-1; Gloor et al. (1986) Cell 47, 687–693; Stone, S.R., Nick, H., Hofsteenge, J., and Monard, D. (1987) Arch. Biochem.Biophys. 252, 237–244) is the only known endogenous thrombin inhibitorpresent in the central nervous system. In vitro, the interplay ofthrombin and PN-1 has been shown to modulate neurite outgrowth ofneuronal cells (Monard, D., Niday, E., Limat, A., and Solomon, F. (1983)Prog. Brain. Res. 58, 359–364; Gurwitz, D. and Cunningham, D. D. (1988)Proc. Natl. Acad. Sci. U. S. A. 85, 3440–3444; Zum et al. (1988) DevNeurosci 10, 17–24; Farmer et al. (1990) Dev. Neurosci. 12:73–80) andthe stellation of astrocytes (Cavanaugh, K. P., Gurwitz, D., Cunningham,D. D., and Bradshaw, R. A. (1990) J. Neurochem. 54, 1735–1743).Furthermore, PN-1 is highly expressed in response to injury of thenervous system (Meier, R., Spreyer, P., Ortmann, R., Harel, A., andMonard, D. (1989) Nature 342, 548–550 25; Hoffmann et al. (1992)Neuroscience 49:397–408). Despite these observations mice lacking PN-1show only subtle phenotypes in the nervous system (Lüthi, A., Putten,H., Botteri, F. M., Mansuy, I. M., Meins, M., Frey, U., Sansig, G.,Portet, C., Schmutz, M., Schroder, M., Nitsch, C., Laurent, J. P., andMonard, D. (1997) J. Neurosci. 17, 4688–4699), suggesting the existenceof an entity that compensates for the lack of PN-1 function in theseanimals.

A 25 kDa carboxypeptidase Y inhibitor, termed Ic or the TFS1 geneproduct, has previously been described in yeast (Bruun, A. W., Svendsen,I., Sorensen, S. O., Kielland-Brandt, M. C., and Winther, J. R. (1998)Biochemistry 37, 3351–3357). Carboxypeptidase Y contains a catalyticSer, His, Asp triad and a trypsin-like oxyanion hole. Its catalyticmechanism is therefore believed to be similar to the serine proteases ofthe trypsin-type (Mortensen, U. H., Olesen, K., and Breddam, K. (1998)in Handbook of proteolytic enzymes (Barrett, A. J., Rawlings, N. D., andWoessner, J. F., eds) pp. 389–393, Academic Press, London). Thesimilarity in sequence between TFS1 (Ic) and 21–23 kDa lipid bindingproteins, such as phosphatidylethanolamine binding protein (PEBP), ledthe authors to speculate that the 21–23 kDa lipid binding proteins mayalso be inhibitors of specific cellular serine carboxypeptidases. Themouse phosphatidylethanolamine-binding protein, PEBP, is made up of 187amino acids and belongs to a family of phospholipid binding proteinsfound in a wide range of species from flowering plants to mammals(Schoentgen, F. and Jolles, P. (1995) FEBS Lett. 369, 22–26). TFS1 andmouse PEBP share 31% identity at the amino acid level.

The phosphatidylethanolamine-binding protein was originally purifiedfrom bovine brain and described as a soluble 23 kDa basic cytosolicprotein (Bernier, I. and Jolles, P. (1984) Biochim. Biophys. Acta 790,174–181). Binding studies revealed its affinity forphosphatidylethanolamine (Bernier, I., Tresca, J. P., and Jolles, P.(1986) Biochim. Biophys. Acta 871, 19–23), nucleotides like GTP and GDPand small GTP-binding proteins and other hydrophobic ligands (Bucquoy,S., Jolles, P., and Schoentgen, F. (1994) Eur. J. Biochem. 225,1203–1210). Independently, PEBP was purified from human brain asneuropolypeptide h3 (Bollengier, F. and Mahler, A. (1980) Neuropeptides1, 119–135 26): sequencing of this protein (Seddiqi, N., Bollengier, F.,Alliel, P. M., Perin, J. P., Bonnet, F., Bucquoy, S., Jolles, P., andSchoentgen, F. (1994) J. Mol. Evol. 39, 655–660) showed 95% amino acidsequence identity with the sequence for the bovine PEBP (Schoentgen, F.,Saccoccio, F., Jolles, J., Bernier, I., and Jolles, P. (1987) Eur. J.Biochem. 166, 333–338). Today members of this family have beenidentified in other mammals including rat (Grandy, D. K., Hanneman, E.,Bunzow, J., Shih, M., Machida, C. A., Bidlack, J. M., and Civelli, O.(1990) Mol. Endocrinol. 4, 1370–1376), mouse (Araki, Y., Vierula, M. E.,Rankin, T. L., Tulsiani, D. R., and Orgebin-Crist, M. C. (1992) Biol.Reprod. 47, 832–843) and monkey (Perry, A. C., Hall, L., Bell, A. E.,and Jones, R. (1994) Biochem. J. 301 (Pt 1), 235–242). Other members ofthe PEBP family are the putative odorant-binding protein in Drosphila(Pikielny, C. W., Hasan, G., Rouyer, F., and Rosbash, M. (1994) Neuron12, 35–49), the putative PEBP of the malaria parasite Plasmodiumfalciparum (Trottein, F. and Cowman, A. F. (1995) Mol Biochem.Parasitol. 70, 235–239), the Ov-16 antigen of Onchocera volvulus (Lobos,E., Altmann, M., Mengod, G., Weiss, N., Rudin, W., and Karam, M. (1990)Mol. Biochem. Parasitol. 39, 135–145) and the toxocaraexcretory-secretory antigen 26 of Toxocara canis (Gems, D., Ferguson, C.J., Robertson, B. D., Nieves, R., Page, A. P., Blaxter, M. L., andMaizels, R. M. (1995) J. Biol. Chem. 270, 18517–18522), two parasiticnematodes. A dosage-dependent suppressor of CDC25 mutations inSaccharomyces cerevisiae, twenty five suppressor 1 (TFS1, Robinson, L.C. and Tatchell, K. (1991) Mol. Gen. Genet. 230, 241–250), and severalproteins in flowering plants (Arabidopsis thaliana (Ohshima, S., Murata,M., Sakamoto, W., Ogura, Y., and Motoyoshi, F. (1997) Mol. Gen. Genet.254, 186–194 27; Kardailsky, I., Shukla, V. K., Ahn, J. H., Dagenais,N., Christensen, S. K., Nguyen, J. T., Chory, J., Harrison, M. J., andWeigel, D. (1999) Science 286, 1962–1965; Kobayashi, Y., Kaya, H., Goto,K., Iwabuchi, M., and Araki, T. (1999) Science 286, 1960–1962) andAntirrhinum (Bradley, D., Carpenter, R., Copsey, L., Vincent, C.,Rothstein, S., and Coen, E. (1996) Nature 379, 791–797)) are alsoincluded in this family.

Despite this widespread expression and the resolution of the 3Dstructures of bovine and human PEBP by X-ray crystallography (Serre, L.,Vallee, B., Bureaud, N., Schoentgen, F., and Zelwer, C. (1998) Structure6, 1255–1265; Banfield, M. J., Barker, J. J., Perry, A. C., and Brady,R. L. (1998) Structure 6, 1245–1254) very little is known about thefunction or properties of the proteins in this family. In rathippocampus, PEBP was described to be the precursor of the hippocampalneurostimulating peptide (HCNP), an undecapeptide that is involved inthe differentiation of neurons in the medial septal nucleus by enhancingthe synthesis of choline acetyltransferase (Ojika, K., Mitake, S.,Tohdoh, N., Appel, S. H., Otsuka, Y., Katada, E., and Matsukawa, N.(2000) Prog. Neurobiol. 60, 37–83). However, the widespread expressionof PEBP outside of the central nervous system including spleen, testis,ovary, muscle and stomach (Bollengier, F. and Mahler, A. (1988) J.Neurochem. 50, 1210–1214; Frayne, J., Ingram, C., Love, S., and Hall, L.(1999) Cell. Tissue Res. 298, 415–423) suggests additional roles forthis protein.

The members of the PEBP family are often highly expressed in growing orelongated cells such as oligodendrocytes, spermatides, and theinflorescence meristem of flowering plants. Together, this expressionpattern and the binding to phospholipids located mainly on the innerleaflet of the plasma membrane suggests a role for PEBP in theorganization of the plasma membrane during cell growth and development.PEBP was reported to be one of several cellular proteins present insidethe human immunodeficiency virus type 1 (HIV-1) virions (Ott, D. E.,Coren, L. V., Johnson, D. G., Kane, B. P., Sowder, R. C., Kim, Y. D.,Fisher, R. J., Zhou, X. Z., Lu, K. P., and Henderson, L. E. (2000)Virology 266, 42–51), supporting a possible role for PEBP in membraneorganization.

On the other hand, the interaction between PEBP and small-GTP bindingproteins leads to speculation that PEBP could be involved in thesignaling machinery. Recently, PEBP was described as a Raf-1 kinaseinhibitor protein (RKIP) that suppresses the MAP-kinase signaling(Yeung, K., Seitz, T., Li, S., Janosch, P., McFerran, B., Kaiser, C.,Fee, F., Katsanakis, K. D., Rose, D. W., Mischak, H., Sedivy, J. M., andKolch, W. (1999) Nature 401, 173–177; Yeung, K., Janosch, P., McFerr n,B., Rose, D. W., Mischak, H., Sedivy, J. M., and Kolch, W.(2000)Mol.Cell. Biol.20, 3079–3085). The authors of this study suggest that PEBPregulates the ERK pathway at the Raf/MEK interface by binding to Raf-1and MEK thereby leading to a competitive inhibition of the ERK pathway.They further showed that the binding of PEBP/RKIP to Raf-1 decreasesduring mitogenic stimulation.

In summary, although possible PEBP functions have been suggested in theart, there has been no definitive demonstration of PEBP proteaseinhibitory activity. PEBP shares no significant homology with otherknown classes of serine protease inhibitors such as the serpins, theKunitz, the Kazal, or the Bowman-Birk family (Barrett, A. J. andSalvesen, G. (1986) Proteinase inhibitors, Elsevier, Amsterdam 28).

SUMMARY OF THE INVENTION

According to the present invention, we provide a method of inhibiting aprotease, preferably a serine protease selected from the groupconsisting of thrombin, chymotrypsin and neuropsin, by contacting theprotease with an effective amount of a phosphoethanolamine bindingprotein (PEBP) family member, preferably PEBP.

The present invention also provides a screening method for identifyingcompounds capable of enhancing or inhibiting a biological activity of aPEBP family member, which involves contacting a protease which isinhibited by a PEBP family member with a candidate compound in thepresence of a partially inhibitory amount of a PEBP family member,assaying proteolytic activity of the protease on a susceptible substratein the presence of the candidate compound and partially inhibitoryamount of the PEBP family member, and comparing the proteolytic activityto a standard level of activity, the standard being assayed when contactis made between the protease and its substrate in the presence of thepartially inhibitory amount of a PEBP family member and the absence ofthe candidate compound. In this assay, an increase in inhibition ofproteolytic activity over the standard indicates that the candidatecompound is an agonist of PEBP inhibitory activity and a decrease ininhibition of proteolytic activity compared to the standard indicatesthat the compound is an antagonist of PEBP inhibitory activity.

In another aspect, a screening assay for agonists and antagonists isprovided which involves determining the effect a candidate compound hason binding of the PEBP family member to a susceptible protease,preferably to its active site. In particular, the method involvescontacting the PEBP-susceptible protease with a PEBP family member and acandidate compound and determining whether binding of the PEBP familymember to the PEBP-susceptible protease is increased or decreased due tothe presence of the candidate compound.

Kits comprising a phosphoethanolamine binding protein (PEBP) familymember for use in any the methods of the invention are also provided.

The present invention also provides a method of inhibiting bloodcoagulation, comprising contacting blood with a phosphoethanolaminebinding protein (PEBP) family member in an amount sufficient to inhibitblood coagulation. In this regard, receptacles and other solid surfacesthat come into contact with blood can be treated with a PEBP familymember, preferably PEBP, to inhibit blood coagulation. In addition,methods for treating anti-thrombogenic diseases or disorders areprovided using a PEBP family member alone or in combination with othermedicaments, such as an anti-coagulant.

In a further aspect of the invention, a method is provided for treatinga disorder or disease, or predisposition thereto, characterized by anincrease in the activity of a protease susceptible to inhibition by aPEBP family member, by administering an effective amount of a PEBPfamily member or an agonist thereof to an individual. Such disordersinclude thrombogenic disorders, neurodegenerative disorder, and cancer.Thus, also provided are compositions comprising a PEBP family member anda pharmaceutically appropriate carrier, such as liposomes foradministration to an individual in need of treatment.

Methods of treatment of diseases, particularly cognitive disorders areprovided and are effected by administering an effective amount of thepharmaceutical compositions. In particular, methods of treating apatient suffering from a neurodegenerative disease selected from amongAlzheimer's disease, cognition deficits, Down's Syndrome, Parkinson'sdisease, cerebral hemorrhage with amyloidosis, dementia pugilistica,head trauma and any disorder characterized by an increase in proteaseactivity, in particular thrombin, neuropsin or chymotrypsin activity, inthe brain, by administering to the patient a therapeutically effectiveamount of a PEBP polypeptide.

Also provided is a PEBP family member comprising the sequence providedin FIG. 2 (SEQ ID NO:1).

In a further aspect of the invention, a method is provided fordiagnosing a disorder or disease characterized by an increase in theactivity of a protease susceptible to inhibition by a PEBP familymember, by determining whether the presence of the PEBP family member isreduced in a sample relative to a standard level, the standard levelbeing from a non-afflicted individual.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Thrombin inhibiting activity in brains of wild type andPN-1^((−/−)) mice. Different parts of wild type (Δ, cerebellum; □,cortex; ∘, remaining parts) and PN-1^((−/−)) (▴, cerebellum; ▪, cortex;●, remaining parts) brains were homogenized and their protein contentmeasured. Aliquots with equal protein contents were stepwise diluted andassayed for thrombin inhibition.

FIG. 2 Identification of the inhibitor by NanoESI mass spectrometry. Thetryptic peptides found to be identical to mouse PEBP (SEQ ID NO:1) areshown in bold. The serine at position 116 originally published(Swiss-Prot accession number P70296) was found to be a glycine that isin fact conserved in all known proteins belonging to thephosphatidylethanolamine-binding protein family. This finding wasconfirmed by DNA sequencing of the IMAGE clone No. 1921274 that has asthe 116_(th) codon a GGT instead of the previously published AGT(GenBank accession number U43206).

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have identified a protease inhibitor that is ableto compensate for the lack of the thrombin inhibitor, PN-1, in PN-1(−/−) mice. The protease inhibitor has been identified as a member ofthe phosphatidylethanolamine binding protein (PEBP) family and has beencharacterized as having specific serine protease inhibitory activity, inparticular thrombin, neuropsin and chymotrypsin inhibitory activities.Thus, in one aspect of the invention, a method is provided of inhibitinga protease, preferably a serine protease, more preferably a serineprotease selected from the group consisting of thrombin, chymotrypsinand neuropsin, the method comprising contacting the protease with aneffective amount of a phosphoethanolamine binding protein (PEBP) familymember.

As used herein, the term “PEBP family member” is meant to encompass PEBPhomolgues or fragments, which can be identified as such by comprisingregions of at least 50% identity, preferably at least 70% identity, morepreferably at least 80% identity, most preferably at least 90% identityto the regions spanning amino acid residues 65–86 and 115–128 of humanPEBP as disclosed by Bruun et al. (see FIG. 1C, 3^(rd) sequence therein;SEQ ID NO:2). Other than related mammalian PEBP proteins, other membersof the PEBP family are the putative odorant-binding protein in Drosphila(Pikielny, C. W., Hasan, G., Rouyer, F., and Rosbash, M. (1994) Neuron12, 35–49), the putative PEBP of the malaria parasite Plasmodiumfalciparum (Trottein, F. and Cowman, A. F. (1995) Mol. Biochem.Parasitol. 70, 235–239), the Ov-16 antigen of Onchocera volvulus (Lobos,E., Altmann, M., Mengod, G., Weiss, N., Rudin, W., and Karam, M. (1990)Mol. Biochem. Parasitol. 39, 135–145) and the toxocaraexcretory-secretory antigen 26 of Toxocara canis (Gems, D., Ferguson, C.J., Robertson, B. D., Nieves, R., Page, A. P., Blaxter, M. L., andMaizels, R. M. (1995) J. Biol. Chem. 270, 18517–18522), the TFS-1 geneproduct (TFS1, Robinson, L. C. and Tatchell, K. (1991) Mol. Gen. Genet.230, 241–250), and several PEBPs in flowering plants (Arabidopsisthaliana (Ohshima, S., Murata, M., Sakamoto, W., Ogura, Y., andMotoyoshi, F. (1997) Mol. Gen. Genet. 254, 186–194 27; Kardailsky, I.,Shukla, V. K., Ahn, J. H., Dagenais, N., Christensen, S. K., Nguyen, J.T., Chory, J., Harrison, M. J., and Weigel, D. (1999) Science 286,1962–1965; Kobayashi, Y., Kaya, H., Goto, K., Iwabuchi, M., and Araki,T. (1999) Science 286, 1960–1962) and Antirrhinum (Bradley, D.,Carpenter, R., Copsey, L., Vincent, C., Rothstein, S., and Coen, E.(1996) Nature 379, 791–797)).

Preferred members are mammalian PEBPs or synthetic PEBPs derived fromthe mammalian sequences, which include amino acid substitutions,deletions and additions compared to naturally occurring PEBPs. Naturallyoccurring PEBPs also include “allelic variants” that are alternate formsof a gene occupying a given locus on a chromosome of an organism. GenesII, Lewin, B., ed., John Wiley & Sons, New York (1985). Most preferredare human PEBP (SEQ ID NO:2) or PEBP encoded by the sequence provided inFIG. 2 (SEQ ID NO:1). Thus, in one aspect of the invention, a PEBPpolypeptide encoded by the sequence provided in FIG. 2 (SEQ ID NO:1) isprovided.

Although the specific examples provided herein are limited to knownmembers of the PEBP family, it will be apparent to one of ordinary skillin the art that PEBP family members yet to be identified, or fragmentsthereof, are easily prepared in light of the teachings of the presentspecification. For example, oligonucleotide templates for thePCR-amplification of the coding sequences of the different PEBPpolypeptides can be modified for use according to the extent of homologybetween the known and new PEBP family members and depending onhybridization conditions. Alternatively, if sufficient identity existsbetween the two sequences, the same template can be used without furthermodification. Alternatively, suitable pairs of oligonucleotide templatescan be used. In addition, variants of the naturally occurring sequenceis foreseen, in particular conservative substitutions of amino acids notessential for protease inhibitory activity.

The PEBP family member can be prepared as a fusion protein to facilitatepurification or certain assay formats. The fusion protein may comprise,in addition to PEBP family member sequences, tags or a reportermolecule, for example. The tag can be, for example, hemagglutinin (HA),repetitive histidine residues (His6) or the like. The reporter molecule(i.e., a signal generating molecule) can be any molecule capable ofproviding a detectable change. Such reporter molecules includefluorescent moieties (e.g., fluorescent proteins or chemical fluorescentlabels), radioactive moieties, phosphorescent moieties, antigens,reporter enzymes and the like. Preferably, the reporter molecule is areporter enzyme whose activity brings about a detectable change. Suchreporter enzymes include, but are not limited to, the following:beta-galactosidase, glucosidases, chloramphenicol acetyltransferase(CAT), glucoronidases, luciferase, peroxidases, phosphatases,oxidoreductases, dehydrogenases, transferases, isomerases, kinases,reductases, deaminases, catalases and urease. The selection of anappropriate reporter molecule will be readily apparent to those skilledin the art.

The PEBP family member or fusion protein can be easily prepared byrecombinant or chemical methods, as well as by using standard proteinpurification techniques, as is apparent to one of ordinary skill in theart. For example, the DNA coding for the PEBP family member or fusionprotein may be comprised in a nucleic acid expression cassettecomprising a promoter operably linked to the nucleic acid encoding thePEBP family member or fusion protein and optionally to transcriptiontermination signals. The fused polypeptides of the fusion protein may beconnected directly or by a spacer

Proteins may also be obtained by synthetic means rather than derivedfrom natural sources, using commercially available protein synthesisersor even ordered from a commercial peptide synthesis service. Synthesizedproteins may comprise any desired sequence modifications, including theuse of altered amino acid residues or the addition of heterologousgroups or side-chains to the polypeptide, and incorporation of labels ortags. Chemically synthesized proteins may also include non-peptidyllinkages as is apparent to those skilled in the art.

The term “effective amount” as used herein with respect to proteaseinhibition means sufficient to inhibit the susceptible protease by atleast 20%, preferably at least 40%, more preferably at least 50% ormore.

The protease inhibitory activities of PEBP family members are useful forprotein purification, in particular proteinase purification (e.g.,thrombin, chymotrypsin and neuropsin), as well as for other productionand research applications. Thus, PEBP family members may be packaged asarticles of manufacture containing packaging material, an acceptablecomposition comprising a PEBP polypeptide, which is effective for thedesired purpose, and a label that indicates that the composition is usedas a protease inhibitor, in particular an inhibitor of thrombin,chymotrypsin and neuropsin.

In one aspect of the invention, screening methods are provided where aprotease susceptible to inhibition by a PEBP family member is contactedwith a PEBP family member and an additional potential proteasemodulator. A change in the level of protease activity detected in thepresence of the potential protease modulator as compared to when saidpotential protease modulator is absent indicates the presence of aprotease modulator. The term “protease susceptible to inhibition by aPEBP family member” as used herein is meant to encompass any proteaseinhibited by a PEBP family member, preferably by a mammalian PEBP. Mostpreferably, the protease is thrombin, chymotrypsin or neuropsin.

In particular, a method is provided for identifying modulators of abiological activity of a PEBP family member, the method comprisingcontacting a protease susceptible to inhibition by a PEBP family memberwith a candidate compound in the presence of a partially inhibitoryamount of said PEBP family member, assaying proteolytic activity of saidprotease in the presence of a susceptible substrate, said candidatecompound and partially inhibitory amount of said PEBP family member, andcomparing the proteolytic activity to a standard level of activity, thestandard level of activity being assayed when contact is made betweenthe protease and said substrate in the presence of the partiallyinhibitory amount of PEBP family member and the absence of saidcandidate compound. An increase in inhibition of proteolytic activityover the standard is correlated with the presence of an agonist of PEBPinhibitory activity, whereas a decrease in inhibition of proteolyticactivity compared to the standard is correlated with the presence of anantagonist of PEBP inhibitory activity. Therefore, an agonist in thepresent context is a compound which increases the natural biologicalfunctions of a PEBP family member or which functions in a manner similarto a PEBP family member, while antagonists decrease or eliminate suchfunctions.

The invention also provides a method of screening compounds to identifythose that enhance or block the action of PEBP family members onproteases, based on their ability to interact with proteases, inparticular with thrombin, chymotrypsin and neuropsin. Thus, a method isprovided for identifying modulators of a biological activity of a PEBPfamily member by contacting a PEBP-susceptible protease, preferablythrombin, chymotrypsin or neuropsin, with a PEBP polypeptide and acandidate compound, and determining whether binding of the PEBP familymember to said PEBP-susceptible protease is increased or decreased dueto the presence of the candidate compound.

For example, a cellular extract or fraction may be prepared from a cellthat expresses or comprises a molecule that binds a PEBP family member,such as a molecule of a signaling or regulatory pathway orphosphatidylethanolamine, or a body fluid can be used in the screeningassays described herein. The preparation can be incubated with a labeledPEBP family member in the absence or the presence of a candidatemolecule that may be a PEBP agonist or antagonist. The ability of thecandidate molecule to bind the binding molecule is reflected indecreased binding of the labeled ligand. Molecules that bindgratuitously, i.e., without inducing the effects of PEBP on binding thePEBP-binding molecule, are most likely to be good antagonists. Moleculesthat bind well and elicit effects that are the same as or closelyrelated to PEBP are agonists.

Another example of an assay for PEBP antagonists is a competitive assaythat combines a PEBP family member and a potential antagonist of aPEBP-susceptible protease, particularly thrombin, chymotrypsin orneuropsin under appropriate conditions for a competitive inhibitionassay. The PEBP family member can be labeled, such as by radioactivity,such that the number of PEBP family member molecules bound to proteasemolecules can be determined accurately to assess the effectiveness ofthe potential antagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a PEBP family member andthereby inhibit or eliminate its activity. Potential antagonists alsomay be small organic molecules, a peptide, a polypeptide such as aclosely related protein or antibody that binds the same sites on abinding molecule, such as PEBP-susceptible protease molecule, withoutinducing PEBP-induced activities, thereby preventing the action of PEBPby excluding PEBP from binding.

Once the potential agonist/antagonist is identified, further analysiscan be carried out to confirm the identification. Such tests are knownin the art and include neurite outgrowth assays, phosphoinositidehydrolysis assays, Ca²⁺ efflux assays, and platelet aggregation assays.

Other potential antagonists include antisense molecules. Antisensetechnology can be used to control gene expression through antisense DNA,or RNA or through triple-helix formation. Antisense techniques arediscussed, for example, in Okano, J. Neurochem. 56: 560 (1991);“Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression”, CRCPress, Boca Raton, Fla. (1988). Triple helix formation is discussed in,for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooneyet al., Science 241: 456 (1988); and Dervan et al., Science 251:136((1991). The methods are based on binding of a polynucleotide to acomplementary DNA or RNA. For example, the 5′ coding portion of apolynucleotide that encodes the mature PEBP family member may be used todesign an antisense RNA oligonucleotide of from about 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription thereby preventingtranscription and the production of PEBP. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into PEBP polypeptide. The oligonucleotides describedabove can also be delivered to cells such that the antisense RNA or DNAmay be expressed in vivo to inhibit production of PEBP.

The agonists and antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as described below.

Serine proteases play a role in the pathology of a variety of disorders,including cerebral ischaemia, neurodegenerative disorders, cataract,myocardial ischaemia, muscular dystrophy and platelet aggregation. Thus,compounds that have activity as serine protease inhibitors areconsidered by those of skill in this art to be useful. For example,thrombin is a key protease in thrombogenesis. Intravascular clottingoccurs frequently with shock, sepsis, cancer, obstetric complications,bums, and liver disease. Thus a thrombin inhibitor is particularlyuseful in inhibiting blood coagulation or preventing embolies.

In one aspect of the invention, therefore, a method is provided forinhibiting or delaying blood clotting comprising contacting blood with aPEBP family member or agonist, preferably a mammalian or human PEBP, inan amount sufficient to inhibit blood coagulation.

Blood generally clots in vitro in four to eight minutes when placed in aglass tube. Clotting is prevented if a chelating agent such asethylenediaminetetraacetic acid (EDTA) or citrate is added to bind Ca²⁺.The ability of a PEBP family member to delay blood clotting can be shownby addition of approprate amounts of the PEBP family member to blood toinhibit blood coagulation completely or delay blood coagulation beyond 8minutes, as desired. The required amounts can be determined empirically.

Thrombotic disorders, including acute vascular diseases, such asmyocardial infarction, stroke, peripheral arterial occlusion, deep veinthrombosis, pulmonary embolism, and other blood system thromboses,constitute major health risks. Such disorders are caused by eitherpartial or total occlusion of a blood vessel by a blood clot, whichconsists of fibrin and platelet aggregates. Therapeutic interventionwith agents that prevent or delay clot formation (i.e., anticoagulants)or with agents that dissolve blood clots (i.e., thrombolytics) is commonpractice. The unexpected anti-thrombin properties of PEBP family memberscan now be applied in treating such thrombotic disorders. Theadministered PEBP family member present in the blood during clotformation delays clotting time and/or may change the character of theclot that is formed to a looser, less stable clot.

The term “thrombotic disorder” as used herein encompasses conditionsassociated with or resulting from thrombosis or a tendency towardsthrombosis. These conditions include conditions associated with arterialthrombosis, such as coronary artery thrombosis and resulting myocardialinfarction, cerebral artery thrombosis or intracardiac thrombosis (dueto, e.g., atrial fibrillation) and resulting stroke, and otherperipheral arterial thrombosis and occlusion; conditions associated withvenous thrombosis, such as deep venous thrombosis and pulmonaryembolism; conditions associated with exposure of the patient's blood toa foreign or injured tissue surface, including diseased heart valves,mechanical heart valves, vascular grafts, and other extracorporealdevices such as intravascular cannulas, vascular access shunts inhemodialysis patients, hemodialysis machines and cardiopulmonary bypassmachines; and conditions associated with coagulapathies, such ashypercoagulability and disseminated intravascular coagulopathy that arenot the result of an endotoxin-initiated coagulation cascade.

The PEBP family member can be administered together with conventionalantithrombotic agents, such as antiplatelet agents, anticoagulant agents(heparin), vitamin K antagonists (coumarin derivatives, warfarin) andthrombolytic agents, in dosages and by routes consistent with the usualclinical practice. [See, generally, Goodman & Gilman, ThePharmacological Basis of Therapeutics, 9th ed., McGraw-Hill, N.Y.(1996).]

Anti-thrombotic agents are also used routinely to prevent the occlusionof extracorporeal devices: intravascular cannulas, vascular accessshunts in hemodialysis patients, hemodialysis machines, andcardiopulmonary bypass machines. Thus, in a further aspect of theinvention, a PEBP family member is coated onto a solid surface,including without limitation those listed above, prior to contacting thesurface with blood.

Serine proteases are known to have an effect on many other disorders andtherefore, PEBP family members are useful in treating an individual inneed of an increased level of PEBP activity (or of decreased proteolyticactivity of a PEBP-susceptible protease, particularly thrombin,chymotrypsin and neuropsin) comprising administering to such anindividual a pharmaceutical composition comprising an amount of a PEBPfamily member, particularly a mature form of the PEBP family member,effective to increase the PEBP activity level (and, thereby decrease thePEBP-susceptible protease activity) in such an individual. Such anindividual may be suffering from a particular disorder or disease ormerely at high risk of obtaining the disorder or disease.

The disorders or diseases include, but are not limited to: Alzheimer'sdisease, cognition deficits, Down's Syndrome, Parkinson's disease,Huntington's disease, and other chronic neurodegenerative diseases,cerebral hemorrhage with amyloidosis, dementia pugilistica, head traumaand conditions characterized by a degradation of the neuronalcytoskeleton resulting from a thrombolytic or hemorrhagic stroke,pulmonary emphysema, arthritis, multiple sclerosis, periodontal disease,cystic fibrosis, respiratory disease, thrombosis, cancer, cachexia,angina, glaucoma, inflamatory disorders, osteoporosis, cardiovasculardisorders such as hypertension, atherosclerotic disorders such ascardiac infarction, and stroke, asthma, psoriasis, demyelinatingdiseases, AIDS immune deficiency, disorders of photoreceptordegeneration, and lens cataract formation, organ transplant rejection,restenosis, muscular dystrophy, renal failure, cerebral vasospasm,pancreatitis, disorders of mitogen induced cell growth and diabeticnephropathy.

As used herein, an effective amount of a compound for treating adisorder is an amount that is sufficient to ameliorate, or in somemanner reduce a symptom or stop or reverse progression of a condition.Such amount may be administered as a single dosage or may beadministered according to a regimen, whereby it is effective.“Treatment” as used herein covers any treatment of a disease in amammal, particular a human, and includes:

-   (a) preventing the disease/condition or symptom from occurring in a    subject which may be predisposed to the disease/condition or symptom    but has not yet been diagnosed as having it;-   (b) inhibiting the disease/condition or symptom, i.e., arresting its    development; or-   (c) relieving the disease/condition symptom, i.e., causing    regression of the disease/condition.

A PEBP family member has now been shown to exhibit selective inhibitionof thrombin, chymotrypsin and neuropsin. In addition, in vitro enzymaticactivity has been demonstrated for recombinantly-expressed purifiedprotein. See Examples 4 and 5. As noted above, PEBP compensates for thelack of PN-1 in PN-1 (−/−) mice. Protease nexin I (PN-1 or glia-derivednexin, GDN) has been shown to inhibit thrombin specifically and topromote, in vitro, neurite extension in neuroblastoma cell lines as wellas primary hippocampal, and sympathetic neurons. The PN-1 gene isinduced transcriptionally and protein levels are increased following ratsciatic nerve axotomy. Treatment of chick developing motoneurons, i.e.E6–E9 lumbrosacral motoneurons that normally undergo apoptosis, withPN-1 results in increased survival of motoneurons. Motoneuron deathexperimentally induced by sciatic nerve lesioning in mouse is alsodecreased by PN-1 addition. Alzheimer-diseased brain regions containhigher PN-1/thrombin complexes compared with free PNI than do normalbrains suggesting that PN-1 may have a role in CNS pathology.Chymotrypsin has also been linked to Alzheimer's disease, and thelocalization of neuropsin to brain tissue also suggests a neurologicalfunction of these proteins.

Thus, due to the functional similarities and tissue localization betweenPEBP and PN-1, PEBP family members can be used for treatingneurodegenerative disorders and peripheral neuropathies such asamyotrophic lateral sclerosis (ALS) or multiple sclerosis. Motoneuron orsensory neuron damage resulting from spinal cord injury also my beprevented by treatment with a PEBP family member. In addition, centralnervous system diseases like Alzheimer's disease may be treated with aPEBP family member or, a small molecule agonist capable of crossing theblood-brain barrier, which agonist can be identified according to themethods of the present invention. For some treatments, it isadvantageous to have the PEBP family member or agonist cross theblood-brain barrier. PEBP's ability to bind phosphatidylethanolaminefacilitates liposome delivery across the blood-brain barrier.

Aside from the nervous system-related disorders described above, PN-1has been shown to inhibit breakdown of extracellular matrix in afibroblast tumor cell line. Such breakdown is thought to enable tumorcells to metastasize by weakening of extracellular matrix which normallyprevents penetration of unrelated cells through a tissue. Thus, a PEBPfamily member may also be used to inhibit extracellular matrixdestruction, in particular that associated with tumors secreting aPEBP-susceptible protease, for instance, neural tissue tumors secretingthrombin, chymotrypsin or neuropsin.

The composition comprising the PEBP family member will be formulated anddosed in a fashion consistent with good medical practice, taking intoaccount the clinical condition of the individual patient (especially theside effects of treatment with PEBP family member alone), the site ofdelivery of the composition, the method of administration, thescheduling of administration, and other factors known to practitioners.The “effective amount” of PEBP family member for purposes herein is thusdetermined by such considerations.

As a general proposition, the total pharmaceutically effective amount ofa PEBP family member administered parenterally per dose will be in therange of about 1 μg/kg/day to 10 mg/kg/day of patient body weight,although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day. If givencontinuously, the PEBP family member is typically administered at a doserate of about 1 μg/kg/hour to about 50 μg/kg/hour, either by 1–4injections per day or by continuous subcutaneous infusions, for example,using a mini-pump. An intravenous bag solution may also be employed. Thelength of treatment needed to observe changes and the interval followingtreatment for responses to occur appears to vary depending on thedesired effect.

Pharmaceutical compositions containing the PEBP family member may beadministered peridurally, orally, rectally, parenterally,intravaginally, intraperitoneally, topically (as by powders, ointments,drops or transdermal patch), bucally, or as an oral or nasal spray. By“pharmaceutically acceptable carrier” is meant a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The term “parenteral” as used hereinrefers to modes of administration that include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

The PEBP family member is also suitably administered bysustained-release systems. Suitable examples of sustained-releasecompositions include semi-permeable polymer matrices in the form ofshaped articles, e.g., films, or mirocapsules. Liposomes comprising thePEBP family member are prepared by methods known in the art: DE3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688–3692(1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030–4034 (1980);EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat.Appl. 83–118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Ordinarily, the liposomes are of the small (about 200–800 Angstroms)unilamellar type in which the lipid content is greater than about 30mol. percent cholesterol, the selected proportion being adjusted for theoptimal PEBP therapy.

Generally, the formulations are prepared by contacting the PEBP familymember (and, optionally, any cofactor which may enhance its activity)uniformly and intimately with liquid carriers or finely divided solidcarriers or both. Then, if necessary, the product is shaped into thedesired formulation. Preferably the carrier is a parenteral carrier,more preferably a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, and dextrose solution. Non-aqueous vehicles such asfixed oils and ethyl oleate are also useful herein, as well asliposomes.

The PEBP family member is typically formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1–10 mg/ml, ata pH of about 3 to 8. It will be understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers will result in theformation of salts.

The invention also provides a composition comprising a PEBP familymember and a pharmaceutically appropriate carrier, such as describedabove, as well as pharmaceutical packs or kits comprising one or morecontainers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. Such kits canoptionally comprise equipment useful in administering the compositionsuch as an inhaler, syringes and the like.

In addition, the PEBP family members may be employed in conjunction withother therapeutic compounds. Agonists of PEBP may be employed in placeof or in addition to a PEBP family member, for instance, for treatingany of the disordersor diseases referred to above.

Similarly, antagonists of PEBP family members, such as anti-PEBPantibodies, may be used in a method for treating an individual in needof a decreased level of PEBP activity in the body (i.e., less inhibitionof a protease susceptible to PEBP) comprising administering to such anindividual a composition comprising a therapeutically effective amountof a PEBP antagonist. As noted above, elimination of PN-1 (describedabove) by homologous recombination leads to reduced long-termpotentiation (LTP) of learning, whereas overexpression of PN-1 resultsin enhanced LTP of hippocampal neurons. Id. Similarly, antagonists PEBPactivity capable of passing the blood-brain barrier can be used toenhance LTP of hippocampal neurons in nervous system conditionscharacterized by excessive PEBP expression.

As stated above, the present inventors have discovered that PEBP is ableto compensate for PN-1 activity in brain, and further that PEBP inhibitsserine proteases, such as thrombin, chymotrypsin and neuropsin. For anumber of disorders, in particular of the central or peripheral nervoussystem and thrombogenesis, significantly higher or lower levels of PEBPprotease inhibitory activity may be detected in certain tissues (e.g.,central and peripheral nervous system tissue and testis) or bodilyfluids (e.g., blood, serum, plasma, urine, seminal fluid, synovial fluidor cerebrospinal fluid) taken from an individual having such a disorder,relative to a “standard” PEBP protease inhibitory activity, i.e., thePEBP protease inhibitory activity level in healthy tissue from anindividual not having the nervous system disorder. Thus, the inventionprovides a diagnostic method useful during diagnosis of nervous systemdisorders, which involves: (a) assaying the PEBP protease inhibitoryactivity level in cells or body fluid of an individual; (b) comparingthe PEBP protease inhibitory activity level with a standard PEBPprotease inhibitory level, whereby an increase or decrease in theassayed PEBP protease inhibitory level compared to the standard level(that from a non-afflicted individual) is indicative of a disorder inthat individual.

By individual is intended mammalian individuals, preferably humans,including adults, children, babies and embryos or fetuses. By “measuringthe level of PEBP” is intended qualitatively or quantitatively measuringor estimating the level of the PEBP activity, PEBP protein amount, orPEBP mRNA in a sample directly (e.g., by determining or estimatingabsolute protein level or mRNA level) or relatively (e.g., by comparingto the PEBP protein level or mRNA level in a second biological sample).Preferably, the PEBP level in the first biological sample is measured orestimated and compared to a standard PEBP level, the standard beingtaken from a second biological sample obtained from an individual nothaving the disorder or being determined by averaging levels from apopulation of individuals not having a disorder. As will be appreciatedin the art, once a standard level is known, it can be used repeatedly asa standard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, body fluid, cell line, tissue culture, or other sourcewhich contains PEBP protein or mRNA. As indicated, biological samplesinclude body fluids (such as sera, plasma, seminal fluid, urine,synovial fluid and cerebrospinal fluid), nervous system tissue, andother tissue sources found to express PEBP. Methods for obtaining tissuebiopsies and body fluids from mammals are well known in the art. Wherethe biological sample is to include mRNA, a tissue biopsy is thepreferred source.

Total cellular RNA can be isolated from a biological sample using anysuitable technique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described inChomczynski and Sacchi, Anal. Biochem. 162:156–159 (1987). Levels ofPEBP mRNA are then assayed using any appropriate method. These includeNorthern blot analysis, S1 nuclease mapping, the polymerase chainreaction (PCR), reverse transcription in combination with the polymerasechain reaction (RT-PCR), and reverse transcription in combination withthe ligase chain reaction (RT-LCR).

Assaying PEBP protein levels in a biological sample can occur using anyart-known method. Preferred for assaying PEBP levels in a biologicalsample are antibody-based techniques or activity-based techniques. Forexample, PEBP expression in tissues can be studied with classicalimmunohistological methods. In these, the specific recognition isprovided by the primary antibody (polyclonal or monoclonal) but thesecondary detection system can utilize fluorescent, enzyme, or otherconjugated secondary antibodies. As a result, an immunohistologicalstaining of tissue section for pathological examination is obtained.Tissues can also be extracted, e.g., with urea and neutral detergent,for the liberation of PEBP protein for Western-blot or dot/slot assay,ELISA or RIA assays (Jalkanen, M., et al., J. Cell. Biol. 101:976–985(1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087–3096 (1987)). Inthis technique, which is based on the use of cationic solid phases,quantitation of PEBP protein can be accomplished using isolated PEBPprotein as a standard. This technique can advantageously be applied tobody fluids. PEBP-protein specific antibodies for use in the presentinvention can be prepared using routine procedures.

Preferred are assays of PEBP activity as described above and exemplifiedbelow. The examples below are provided solely for illustrative purposesand are not to be found limiting to the appended claims.

EXAMPLES Example 1 Detection of Thrombin Inhibiting Activity inPN-1^((−/−)) Brains

Although PN-1 is thought to play an important role in the centralnervous system, surprisingly, mice lacking PN-1 show only a subtlephenotype in the nervous system, indicating the potential existence of amolecule that compensates for the lack of PN-1 function in theseanimals. This example demonstrates that a serine protease inhibitor ispresent in brain homogenates of PN-1^((−/−)), which could function inthe same way as PN-1.

Brain tissue was prepared from wild-type, C57bl/6 mice (BRL) ofdifferent ages or PN-1^((−/−)) mice and tested for protease inhibitoryactivity. Briefly, deeply anesthetized animals were pericardiallyperfused with PBS without Ca²⁺ or Mg²⁺ for 5–10 min to obtain blood-freebrain tissue. The brain tissue was either homogenized as a whole ordivided into tissue of the cerebellum, cortex and remaining parts of thebrain before homogenizing each fraction. The tissue or fraction washomogenized for 40 sec using a Polytron (Kinematica GmbH) in 10 mMHepes, pH 7.5, 0.2% Tween-20, 320 mM sucrose and 1 mM EDTA. Thehomogenates were cleared by centrifugation (12,000×g, 30 min, 4° C.) andthe supernatants filtered through a Millex-HA 0.45 μm filter unit(Amicon).

The resulting supernatants were assayed for the presence of thrombininhibitory activity. Human α-thrombin was prepared as described by Stoneet al. (Stone, S. R. and Hofsteenge, J. (1986) Biochemistry 25,4622–4628) and diluted in enzyme buffer (67 mM Tris, pH 8.0, 133 mMNaCl, 0.13% PEG-6000) to provide 0.005 pmol in the final assay mix.Stepwise dilutions (1:160; 1:80; 1:40; 1:20 and 1:10) of aliquots of thehomogenate supernatants containing equal amounts of proteins (12+/−2mg/ml determined by the Bradford assay) were prepared as samples for theassay. Eighty (80) μl of the samples were mixed with 10 μl thrombin(0.005 pmol) in a 96-well plate and incubated for 30 min at 37° C. Afteraddition of 10 μl chromogenic substrate(H-D-Ile-Pro-Arg-para-nitroanilide, Chromogenix, 1.25 mg/ml in H₂O), anyamidolytic activity was determined by measuring the rate of hydrolysisof the chromogenic substrate at 405 nm over 30 min using a THERMOmaxmicroplate reader (Molecular Devices).

The assay revealed the presence of a thrombin inhibitory activity in allinvestigated brain compartments with essentially equivalent levels ofactivity in the various brain compartments tested here. (See FIG. 1).The presence of a thrombin inhibitor is clearly demonstrated to exist inPN-1^((−/−)) brain tissue, even though thrombin inhibition wasapproximately three times higher in wild type brain tissue than inPN-1^((−/−)) brain tissue. Heat treatment of PN-1^((−/−)) brainhomogenates (95° C. for 5 min) completely abolishes thrombin inhibitionsuggesting that the inhibitory activity is due to a protein.

Example 2 Detection of Thrombin Binding Protein in PN-1^((−/−)) Brains

An electrophoretic mobility shift assay was performed to address thequestion as to whether a complex is formed between thrombin and one ormore components of PN-1^((−/−)) brain tissue. Briefly, aliquots (8 μland 24 μl) of whole brain homogenate supernatant (3.2 μg protein perμl), prepared as described in Example 1 from perfused PN-1^((−/−)) mice,were preincubated with 40 ng human α-thrombin in enzyme buffer in afinal volume of 40 μl for 30 min at 37° C. After the preincubation step,5 μl non-reducing sample buffer (312.5 mM Tris pH6.8, 50% glycerol and0.05% bromophenol blue) were added and without heat denaturation of thesample prior to loading, the proteins were resolved by SDS-PAGE undernon-reducing, semi-native (0.1% SDS) conditions.

The resolved proteins were electroblotted on Protran nitrocellulosetransfer membrane (Schleicher-Schuell) in a trans-blot SD semi-drytransfer cell (BioRad) at 3 mA/cm² for 40 min. and analyzed for highmolecular weight complexes with a polyclonal rabbit anti-human thrombinantibody (American Diagnostics, #4702) as primary antibody and anHRP-coupled donkey anti-rabbit antibody (Amersham) as secondaryantibody. The western blot was visualized using the ECL detection kit(Amersham).

Preincubation of brain homogenates with thrombin in this way results inthe formation of complexes of approximately 60 kDa. This size suggests acomplex between thrombin (37 kDa) and a second protein of approximately23 kDa.

To validate the formation of a complex between thrombin and a potentialinhibitory protein, a co-immunoprecipitation was performed. Briefly, twowhole, wild type mouse brains were homogenized as described inExample 1. One half of the sample was incubated with 60 μl humanα-thrombin (1 nM) in enzyme buffer (67 mM Tris, pH 8.0, 133 mM NaCl,0.13% PEG-6000), the other half with 60 μl enzyme buffer alone (negativecontrol), for 30 min at 37° C. After preincubation, animmunoprecipitation was performed with protein A-Sepharose beads coatedwith the anti-thrombin monoclonal antibody EST-6. Protein A-SepharoseCL-4B beads (Amersham-Pharmacia) were coated with monoclonal antibodiesagainst thrombin, EST-6 (American Diagnostics), as described elsewhere(Harlow, E. and Lane, D. (1988) in Antibodies (Harlow, E. and Lane, D.,eds) pp. 522–523, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.). EST-6 is a monoclonal antibody that recognizes free thrombin aswell as thrombin complexed with inhibitors (e.g. antithrombin-III)(Dawes, J., James, K., Micklem, L. R., Pepper, D. S., and Prowse, C. V.(1984) Thromb. Res. 36, 397–409). Fifty (50) μl of the EST-6 coatedbeads were added to the samples and the mixtures incubated at 4° C.overnight with gentle shaking. The beads were then washed 3 times with 1ml enzyme buffer each, and then the co-immunoprecipitated proteinsrecovered by resuspending the beads in 50 μl sample buffer. Afterdenaturation at 95° C. for 5 min, the bead supernatants were resolved by12.5%-SDS-PAGE. The gel was then silver stained following themanufacture's protocol (BioRad Silver Stain) to visualize the proteins.

As the binding properties between thrombin and the putative inhibitorwere not known initially, the co-immunoprecipitation was performed undervery low stringency conditions resulting in a high background.Nevertheless a protein of approximately 20 kDa could be identified asbeing co-immunoprecipitated after preincubation with human α-thrombinbut not with buffer.

In summary, these data suggested the existence of a thrombin bindingprotein of 20–23 kDa as a potential candidate thrombin inhibitor.

Example 3 Identification of Thrombin Inhibiting Activity of ThrombinBinding Protein in PN-1^((−/−)) Brains

Brain homogenates were fractionated according to their molecular weightsby gel filtration to establish whether the thrombin binding proteinco-elutes with the moiety identified in Example 1 as having thrombininhibitory activity. Brain homogenates (whole) from PN-1^((−/−)) ofdifferent ages (14 days; 6 months; 1 year; 2 years) or wild-type micewere prepared essentially as described in Example 1. The partialpurification of the inhibitor was obtained by applying the homogenate toa Superdex 200 16/60 gel filtration column (Amersham-Pharmacia)equilibrated with 50 mM Tris, pH 8.0, 100 mM NaCl at 4° C. at a flowrate of 0.25 ml/min with the equilibration buffer. Fractions of 0.25 mlwere collected and assayed for thrombin inhibiting activity.

A thrombin inhibitory activity elutes from the column in fractions no.56–63, corresponding to an elution volume of 14–15.75 ml. Calibration ofthe gel filtration column with ribonuclease A (13.7 kDa),chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), and bovine serumalbumin (67 kDa) under the same elution conditions indicated a molecularweight for the inhibitory activity of 21–24 kDa. The amount of the 21–24kDa inhibitory activity is approximately the same in brain homogenatesprepared from mice of different ages (from 14 days to two years) as wellas between wild type and PN-1^((−/−)) mice. A peak in hydrolysis rateswas seen in fractions 64–66 of fractionated brain tissue from olderanimals, probably corresponding to a proteinase having a molecularweight of substantially less than 20 kDa, which is upregulated with age.

Further purification of the inhibitor was pursued to allowidentification of the thrombin inhibitor. In previous experiments, theinhibitor was found not to bind to Q-Sepharose or heparin-Sepharose atpH 9.0 or to SP-Sepharose at pH 5.0. Because of the need to monitoractivity in the flow-through or the eluate of these columns, it was notappropriate to perform the chromatography steps at higher or lower pHvalues. Therefore, a combined anion exchange and heparin affinitychromatography followed by a cation exchange chromatography was used toallow recovery of the inhibitor from the flow-through of the columns.The heparin column was included because of its ability to bind PN-1whose thrombin inhibitory activity is well established (Van Nostrand, W.E., Wagner, S. L., and Cunningham, D. D. (1988) Biochemistry 27,2176–2181; Guenther et al (1985) EMBO J. 4: 1963–1966).

Brain tissue homogenates for ion exchange chromatography were preparedfrom brain tissue of two wild type mice as in Example 1 but using 20 mMethanolamine, pH 9.0, 100 mM NaCl, 0.2% Tween-20, 320 mM sucrose, 1 mMEDTA as homogenization buffer, and cleared of particulate material. Thecleared and filtered homogenates were applied on a 1 ml HiTrapQ-Sepharose column directly connected to a 1 ml HiTrap heparin-Sepharosecolumn (Amersham-Pharmacia) with 20 mM ethanolamine, pH 9.0, 100 mM NaClat a flow rate of 2 ml/min. The flow-through (2 ml) was diluted with 10ml 50 mM Acetate, pH 5.0, 70 mM NaCl and concentrated to 3 ml using acentricon YM-10 (5.000×g, 1 h, 4° C.). After exchanging the buffer witha buffer appropriate for cation exchange chromatography (50 mM Acetate,pH 5, 70 mM NaCl), the sample was loaded on a 1 ml HiTrap SP-Sepharosecolumn (Amersham-Pharmacia) with the same buffer at a flow rate of 2ml/min. The flow-through was concentrated to 300 μl as above. Theconcentrated flow-through of the HiTrap SP-Sepharose purification stepwas applied to a 15%-SDS-PAGE, and silver staining of the gel revealedonly a single band of 20–25 kDa.

The protein separated by SDS-PAGE was excised from the gel, reduced withDTT, alkylated with iodoacetamide and cleaved with trypsin (sequencinggrade, Promega) as described (Shevchenko, A., Wilm, M., Vorm, O., andMann, M. (1996) Anal. Chem. 68, 850–858). The extracted tryptic peptideswere desalted with 5% formic acid, 5% methanol in H₂O on a 1 μl PorosP20 column and concentrated to 1 μl with 5% formic acid, 50% methanol inH₂O directly into the nanoelectrospray ionization (NanoESI) needle.NanoESI mass spectrometry (MS) was performed according to the publishedmethod of Wilm et al. (Wilm, M. and Mann, M. (1996) Anal. Chem. 68,1–8). The mass spectra were acquired on an API 300 mass spectrometer (PESciex) equipped with a NanoESI source (Protana).

Sequence tags from five peptides were obtained that all fitted to theamino acid sequence of the mouse phosphatidylethanolamine-bindingprotein (GenBank accession number P70296), with the exception that theserine at position 116 of the GenBank P70296 sequence was found to be aglycine (SEQ ID NO:1).

Example 4 Recombinant Expression of Mouse PEBP

Recombinant PEBP was prepared to establish that it is able to act as aprotease inhibitor. A cDNA coding for the mouse PEBP was amplified usingPwo DNA polymerase (Roche), the IMAGE clone 1921274 (Sugano mouse,kidney) as template and appropriate primers. Sequencing of the 1921274clone showed that the 3′-end of this cDNA is altered compared to thepublished sequences for the mouse PEBP-mRNA (GenBank accession numberU43206) resulting in a replacement of the last ten amino acid of thePEBP. Consequently we used an antisense primer in the PCR that codes forthe correct last ten amino acids. The cDNA amplified with this primerwas cloned via the Not I and Hind III sites into the pCEP-Pu expressionvector, i.e., pCEP4 (Invitrogen) with a puromycin instead of ahygromycin resistance gene, under the control of a CMV promoter. Thecorrect structure of all constructs was confirmed by DNA sequencing. Thesequencing reactions were performed using Dye Terminators (BigDye, PEBiosystems) with a Perkin-Elmer GeneAmp PCR system 9700 or 2400thermocycler and analyzed on an ABI PRISM 377 DNA sequencer.

The pCEP-Pu-PEBP construct and the empty vector as a negative controlwere transfected into Rat-1 cells (ATCC). Alternative cells may be used,such as COS-7 cells. Rat-1 cells (1.2×10⁵) were plated onto a 60-mm cellculture dish in 2 ml normal growth medium (Dulbecco's modified Eagle'smedium with 10% fetal calf serum). After 24 h, the cells weretransfected with 4 μg pCEP-Pu-PEBP or pCEP-Pu using the FuGENE 6transfection reagent (Roche) according to supplier's instructions. Thegrowth medium was exchanged to DMEM without serum supplemented with 5μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml sodium selenite, 16 μg/mlputrescin, and 10 ng/ml progesterone 24 h after the transfection. Afteranother 48 h the conditioned media were collected and aliquots (5, 10,20, 40 and 80 μl) assayed for thrombin inhibitory activity. The mediumconditioned by the PEBP transfected cells showed a significant increasein inhibition of thrombin compared to the supernatant of the controlcells, with inhibitory activity increasing with increasing amount ofconditioned medium added.

To show that the inhibitory activity detected is indeed due to PEBP,Rat-1 were transfected with pCEP-PU-PEBP-HA. The cDNA coding for themouse PEBP with a C-terminal hemagglutinin-tag (PEBP-HA) was amplifiedfrom the IMAGE clone 1921274 (Sugano mouse, kidney) as described aboveusing appropriate primers introducing an HA tag. The PCR product wascloned via the Not I and Hind III sites into the expression vectorpCEP-Pu under the control of a CMV promoter. The correct structure ofthe construct was confirmed by DNA sequencing as above. Rat-1 cells weretransfected with pCEP-Pu-PEBP-HA (4 μg) and transfected cells grownessentially as described above.

The presence of HA-tagged PEBP was determined in cell lysates as well asthe conditioned medium using an anti-HA antibody. In brief, conditionedmedium was collected and TCA precipitated, whereas cells were lysed inSDS-sample buffer. Twenty (20) μg total protein of the cell lysates orthe TCA-precipitated proteins of 1 ml conditioned medium (dissolved insample buffer) were resolved by 12.5%-SDS-PAGE, electrotransferred to anitrocellulose membrane and detected with an anti-[HA]-peroxidaseconjugate (Roche).

PEBP-HA could be detected in the cell lysate as an approximately 25–30kDa band on the membrane, and to a lesser extent also in the conditionedmedium. Thrombin assays with the lysate and the conditioned medium ofthese cells carried out as described in Example 1 revealed an increasein inhibitory activity as well.

As PEBP is thought to be a cytoplasmic protein and lacks a secretionsignal in its sequence, it was somehow unexpected to detect it in theconditioned medium of PEBP-expressing cells. To validate thisextracellular localization of PEBP, RAT-1 cells were transfected withpCEP-Pu-HA-PEBP-IRES-GFP, a construct encoding HA tagged PEBP fused togreen fluorescent protein (GFP). The cDNA coding for the mouse PEBP witha N-terminal hemagglutinin-tag (HA-PEBP) was amplified essentially asdescribed above using appropriate primers to introduce an N-terminal HAtag. The PCR product was cloned via the Not I and Hind III sites intothe expression vector pCEP-Pu-IRES-GFP under the control of a CMVpromoter. The correct structure of the construct was confirmed by DNAsequencing as above.

Rat-1 cells were transfected with pCEP-Pu-HA-PEBP-IRES-GFP essentiallyas described above. 48 h after transfection the growth medium wasaspirated, the cells were washed 3 times with phosphate buffered saline(PBS), fixed for 20 min. with 4% paraformaldehyde in PBS, and againwashed with PBS. After blocking for 30 min. with 3% BSA in PBS the cellswere incubated with a rhodamine-anti-[HA]-conjugate (Roche) in blockingsolution for 1 hr and washed with PBS. For permeabilization, the cellswere fixed in 4% paraformaldehyde with 15% picric acid, PBS with 0.2%Triton-X 100 was used, and the cells were incubated prior to blocking in1.5 M Tris, pH 8.8, 0.4% SDS.

The Rat-1 cells transfected with an HA-PEBP-IRES-GFP construct displayedHA-PEBP on their surface. Non permeabilized and permeabilized cells wereimmunostained with an anti-[HA]-rhodamine conjugate, and bothnon-permeabilized and permeabilized cells were shown to be stained atthe cell surface and cytoplasm.

The amino acid sequence of PEBP contains no obvious secretion signal andprevious immunohistochemical studies attributed a cytoplasmiclocalization to this protein. The existence of active PEBP in thesupernatant of transfected cells and the immunodetection of HA-taggedPEBP on the cell surface shown here, demonstrate that at least in vitrothe localization of PEBP is not restricted to the cytoplasm or the innerleaflet of the plasma membrane. As HA-positive but non-transfected(GFP-negative) cells were not observed, the HA-immunoreactivity is notdue to binding of HA-PEBP released to the medium by possibly dyingcells.

Example 5 Protease Inhibition Profile of PEBP

To evaluate the inhibitory profile of PEBP, RAT-1 cells were transfectedwith a construct coding for PEBP with a 6×His-tag at the N-terminus. ThecDNA coding for the mouse PEBP was amplified with Pwo DNA polymerase(Roche) from the IMAGE clone 1921274 (Sugano mouse, kidney) usingappropriate primers (5′-CTC TAA GCT TCC ATG GCC GCC GAC ATC-3′, SEQ IDNO:3; and 5′-TCA AAG CGG CCG CTA CTT CCC TGA ACA GCT GCT CGT TAC AGC CTTGGG CAC ATA GTC ATC CCA CTC-3′, SEQ ID NO:4). The PCR product was clonedvia the Not I and Hind III sites into the expression vectors pCEP-Puunder the control of a CMV promoter. A cDNA coding for PEBP with astretch of six histidine residues fused to the carboxy-terminus wasamplified from this construct using the oligonucleotides 5′ CTC TAA GCTTCC ATG GCC GCC GAC ATC-3′, SEQ ID NO:3; and 5′-TCA AAG CGG CCG CTT AATTAA CGT GAT GGT GAT GGT GAT GCT TCC CTG ACA GCT GCT CG-3′; SEQ ID NO:5.The correct structure of all constructs was confirmed by DNA sequencingas above.

Rat-1 cells (4×10⁵) were plated in 10-cm cell culture dishes and after24 h transfected with 16 μg pCEP-Pu-6×His-PEBP or pCEP-Pu essentially asdescribed above in Example 4. After a further 24 h, the medium wasexchanged to serum-free DMEM with supplement. 72 h after transfectionthe medium was collected from four dishes and the cells lysed in 600 μlenzyme buffer (67 mM Tris, pH 8.0, 133 mM NaCl, 0.13% PEG-6000)containing 10 mM imidazole. The recombinant 6×His-PEBP protein waspurified using the Ni-NTA Spin Kit (Qiagen) under native conditionsfollowing the manufacture's protocol with enzyme buffer containing 20 mMimidazole as washing buffer and enzyme buffer containing 250 mMimidazole as elution buffer. The eluate was desalted over a NAP-5 columnto remove the imidazole resulting in a total volume of 1 ml. Cell lysateof “empty” vector transfected cells was treated in the same way andserved as control. Alternatively, 20 mM sodium phosphate pH6.8, 320 mMsucrose, 0.2% TWEEN-20 and 1 mM EDTA can be used as enzyme buffer.

Six different serine proteases were diluted in enzyme buffer (67 mMTris, pH 8.0, 133 mM NaCl, 0.13% PEG-6000) and used in protease activityassays with the following final amounts: thrombin, trypsin andchymotrypsin, 0.005 pmol; t-PA and neuropsin, 0.5 pmol; and pancreaticelastase, 4 pmol. Human α-thrombin was prepared and according to Stoneet al. (Stone, S. R. and Hofsteenge, J. (1986) Biochemistry 25,4622–4628); all other proteases are commercially available (e.g., Sigma,Fluka). Each diluted protease (10 μl) was preincubated with 80 μl of a6×His-PEBP preparation that had been shown to inhibit the amidolyticactivity of 0.005 pmol thrombin to approx. 55%. The pre-incubation wascarried out in a 96-well plate for 30 min at 37° C. After addition of 10μl chromogenic protease substrate (H-D-Ile-Pro-Arg-para-nitroanilide,Chromogenix, 1.25 mg/ml in H₂O for all proteases other thanchymotrypsin; or N-Succinyl-Ala-Ala-Pro-Phe-para-nitroanilide, Sigma,2.3 mg/ml in H₂O for chymotrypsin), any remaining amidolytic activitywas determined by measuring the rate of hydrolysis of the chromogenicsubstrate at 405 nm over 30 min using a THERMOmax microplate reader(Molecular Devices). The 6×His-PEBP sample caused a 25% inhibition of0.005 pmol chymotrypsin and a 70% inhibition of 0.5 pmol neuropsin. Theamidolytic activities of trypsin (0.005 pmol), tissue plasminogenactivator (0.5 pmol), and pancreatic elastase (4 pmol) were notaffected.

To determine the inhibition constants, the rates for the hydrolysis ofthe chromogenic substrates were measured as described above at fixedenzyme concentrations and substrate concentrations ranging from 3.5 μMto 216 μM and PEBP-H6 concentrations between 0 and 2.9 μM. The measuredvalues were fitted with the software GraFit 4.0 (Erithacus Software) tothe equation for a competitive inhibitor. Thrombin and chymotrypsin arecompetitively inhibited by PEBP-H6 with apparent K_(i) of 3.8+/−1.3×10⁻⁷and 1.8+/−1.0×10⁻⁶, respectively.

An interference of the C-terminal histidine tag could be excluded by theuse of purified N-terminal His-tagged PEBP, partially purified PEBP withan N- or C-terminal hemagglutinin tag, as well as the protein encoded bythe IMAGE clone No. 1921274 with 10 different amino acids at theC-terminal. All these different proteins showed similar inhibitoryproperties.

In summary, a serine protease inhibitor from brain tissue, PEBP, isdemonstrated to inhibit the amidolytic activities of thrombin,neuropsin, and chymotrypsin, but not trypsin, tissue-type plasminogenactivator (t-PA), or pancreatic elastase. Data supporting PEBP'sinability to inhibit the serine proteases kallikrein and activatedprotein C were also obtained using partially purified PEBP fractions.

Example 6 Preparation of Antibodies Against PEBP and Localization ofPEBP

In order to raise a polyclonal antiserum against PEBP, two C-terminalpeptides of the mouse PEBP (amino acids 144–159 and 174–187) werecrosslinked to ovalbumin and injected into rabbits following standardprotocols. The specificity of the immune serum was assessed by thedetection of a single 21 kDa band on immunoblots of mouse brainhomogenate.

Rat-1 fibroblasts were washed 3 times with PBS, fixed for 20 min with 4%paraformaldehyde in PBS at room temperature, and again washed with PBS.For permeabilization, the cells were fixed in 4% paraformaldehyde with15% picric acid in PBS (20 min, room temperature) and washed with PBSwith 0.2% Triton-X 100. After blocking for 30 min with 3% BSA in PBS thecells were incubated for one hour with antiserum (1:1500 in blockingsolution) and then washed with PBS. Immunofluorescence detection wasperformed using the Alexa 488 goat anti-rabbit IgG conjugate (MolecularProbes) as secondary antibody. PEBP-immunoreactivity was seen inpermeabilized and non-permeabilized cells. The detection ofPEBP-immunoreactivity at the surface of non-permeabilized Rat-1 cellssupports an extracellular localization of PEBP, in addition to itscytoplasmic localization.

All publications cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

1. A method of inhibiting the protease thrombin, said method comprisingcontacting said protease with an effective amount of aphosphoethanolamine binding protein (PEBP) family member.
 2. The methodof claim 1, wherein said protease is thrombin.
 3. The method of claim 1,wherein said PEBP family member is a mammalian PEBP or a fragmentthereof.
 4. The method of claim 3, wherein said PEBP family member ishuman PEBP.
 5. The method of claim 3, wherein said PEBP is encoded bythe sequence provided in FIG.
 2. 6. The method of claim 1, furthercomprising contacting said protease and protease inhibitor with apotential protease modulator and determining a change in the level ofprotease activity as compared to when said protease inhibitor is absent.